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International Journal for Parasitology: Parasites and Wildlife 5 (2016) 48e55

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International Journal for Parasitology:Parasites and Wildlife

journal homepage: www.elsevier .com/locate/ i jppaw

Lungworm seroprevalence in free-ranging harbour seals andmolecular characterisation of marine mammal MSP

Sophia Arlena Ulrich a, b, Kristina Lehnert a, Ana Rubio-Garcia c,Guillermo J. Sanchez-Contreras c, Christina Strube b, Ursula Siebert a, *

a Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Werftstrasse 6, 25761, Buesum, Germanyb Institute for Parasitology, University of Veterinary Medicine Hannover, Buenteweg 17, 30559, Hannover, Germanyc Seal Rehabilitation and Research Centre, Hoofdstraat 94a, 9968 AG, Pieterburen, The Netherlands

a r t i c l e i n f o

Article history:Received 17 December 2015Received in revised form13 February 2016Accepted 17 February 2016

Keywords:Phoca vitulinaLungworm infectionOtostrongylus circumlitusParafilaroides gymnurusELISAMajor sperm protein

* Corresponding author. Institute for Terrestrial anUniversity of Veterinary Medicine, Foundation, HannBuesum, Germany.

E-mail address: ursula.siebert@tiho-hannover.de (

http://dx.doi.org/10.1016/j.ijppaw.2016.02.0012213-2244/© 2016 The Authors. Published by Elseviercreativecommons.org/licenses/by-nc-nd/4.0/).

a b s t r a c t

Harbour seals (Phoca vitulina) are frequently infected with the lungworms Otostrongylus circumlitus andParafilaroides gymnurus. The infection is often accompanied by secondary bacterial infections and cancause severe bronchopneumonia and even death in affected animals. Hitherto, the detection of lung-worm infections was based on post mortem investigations from animals collected within strandingnetworks and a valid detection method for live free-ranging harbour seals was not available. Recently, anELISA was developed for detecting lungworm antibodies in harbour seal serum, using major spermprotein (MSP) of the bovine lungworm, Dictyocaulus viviparus as recombinant diagnostic antigen. Todetermine lungworm seroprevalence in free-ranging harbour seals, serum was taken from four differentseal age groups (n ¼ 313) resulting in an overall prevalence of 17.9% (18.9% of males, 16.7% of females).0.7% of harbour seals up to six weeks of age were seropositive, as were 89% of seals between six weeksand six months, 53.6% between six and 18 months and 24.2% of seals over 18 months of age. In the 18months and over age group, seropositive animals showed statistically significant reductions in bodyweight (P ¼ 0.003) and length (P < 0.001). Sera from lungworm infected harbour seals in rehabilitation(n ¼ 6) revealed that duration of antibody persistence may be similar to that of lungworm infected cattle,but further studies are needed to confirm this. Phylogenetic analyses of MSP sequences of differentmarine and terrestrial mammal parasitic nematodes revealed that lungworm MSP of the genus Dic-tyocaulus (superfamily Trichostrongyloidea) is more closely related to metastrongylid marine mammallungworms than to trichostrongylid nematodes of terrestrial hosts.© 2016 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is anopen access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

After the first seal epidemic occurred in 1988/89, caused by aphocine distemper virus, a network along the coasts of the GermanFederal State SchleswigeHolstein was established to monitorstranded marine mammals. During necropsy of stranded harbourporpoises and harbour seals findings frequently showed nematodesincluding lungworms Otostrongylus circumlitus (Crenosomatidae)and Parafilaroides gymnurus (Filaroididae) of harbour seals (Lehnertet al., 2005, 2007; Siebert et al., 2007). O. circumlitus was found in

d Aquatic Wildlife Research,over, Werftstrasse 6, 25761,

U. Siebert).

Ltd on behalf of Australian Society f

the bronchi, in the right heart chamber, the Vena pulmonalis and inthe blood vessels of the liver (De Bruyn, 1933; Onderka, 1989;Claussen et al., 1991; Measures, 2001; Lehnert et al., 2007), whileP. gymnurusmainly parasitised the alveoles and bronchioles (Stroudand Dailey, 1978; Claussen et al., 1991). Varying lungworm preva-lence, up to 76%, was reported in harbour seals found in the GermanWadden Sea (Claussen et al., 1991; Lehnert et al., 2007; Siebertet al., 2007). Infections were age-related, with most infectionsoccurring in young animals between two and 18 months of age.Harbour seals may start acquiring lungworm infections afternursing for four weeks and after a post-weaning fast of 15e17 days(Muelbert and Bowen, 1993; Ross et al., 1994) when they start toconsume prey species (Measures, 2001). Benthic fish were identi-fied as potential intermediate hosts of lungworms (Dailey, 1970;Bergeron et al., 1997a; Lehnert et al., 2010); however, the com-plete life cycle is yet unknown.

or Parasitology. This is an open access article under the CC BY-NC-ND license (http://

S.A. Ulrich et al. / International Journal for Parasitology: Parasites and Wildlife 5 (2016) 48e55 49

Lungworms in harbour seals can cause severe pathologicalchanges, like obstruction of bronchial tubes, and are often accom-panied by bacterial infections leading to severe bronchopneumoniaand death (Measures, 2001; Lehnert et al., 2007; Siebert et al.,2007; Rijks et al., 2008). Clinical symptoms include broncho-spasm, anorexia, dehydration, and individual O. circumlitus speci-mens can be observed in sputum (Bergeron et al., 1997a; Measures,2001). Lungworm infections are mainly reported from strandedharbour seals during post mortem examinations (Claussen et al.,1991; Siebert et al., 2007; Rijks et al., 2008), but those data canbe biased by different influences such as age, diseases andanthropogenic activities (Claussen et al., 1991; Measures, 2001;Siebert et al., 2007). Diagnosis in living seals is difficult, as detect-ing lungworm larvae in faeces has limited sensitivity (Schnieder,1992). Collecting harbour seal faeces is logistically challengingand assigning samples to individual free ranging seals is notfeasible. Due to the difficulties in diagnosing lungworm infectionsin living seals, prevalence data in the free-ranging harbour sealpopulations is missing. Therefore, an existing ELISA for immuno-diagnosis of the bovine lungworm Dictyocaulus viviparus(Schnieder, 1992; von Holtum et al., 2008) was adapted to harbourand grey seals with a resulting sensitivity of 98% and a specificity of100% (Ulrich et al., 2015). The ELISA represents a reliablemethod fordiagnosing lungworm antibodies in serum samples of free-rangingharbour seals. Recombinant major sperm protein (MSP), a proteinfamily occurring in nematode sperm only (Klass and Hirsh, 1981;Ward et al., 1988) serves as diagnostic antigen.

Information about the molecular structure of MSP from nema-todes infecting harbour seals and harbour porpoises is missing.Previous phylogenetic analyses within the Metastrongyloidea havebeen performed on the base of large-subunit and small-subunitribosomal (r)RNA (Carreno and Nadler, 2003), the ITS-2 region ofrDNA (Lehnert et al., 2010) and the 18S and 28S rRNA (Chilton et al.,2006). Those analyses confirmed the close relationship of marinemammal lungworms within their superfamily Metastrongyloidea,an evolutionary old group that was derived from the terrestrialancestors of seals and porpoises (Anderson, 1984; Carreno andNadler, 2003).

The aim of this study was to assess lungworm seroprevalence infree-ranging harbour seals in different age groups. Furthermore,consecutive serum samples of harbour seals in rehabilitation wereanalysed to obtain first information on the persistence of serumanti-lungworm-MSP antibodies. Additionally, MSP genes fromdifferent nematodes infecting harbour porpoises and harbour sealswere identified and sequenced to explore phylogenetic relation-ships between marine and terrestrial parasitic nematodes.

2. Material and methods

2.1. ELISA

2.1.1. Age determination of harbour sealsThe approximate age of sampled harbour seals was determined

and sorted in age groups, considering sampling date, body-lengthand body-weight. In young seals, navel and canine developmentwas additionally considered. Age group (AG) 1 included harbourseals from birth to six weeks of age, AG 2 harbour seals from sixweeks to six months, AG 3 from six to 18 months and AG 4 above 18months of age.

2.1.2. Sera of free-ranging harbour sealsAll experimental procedures involving harbour seals were

approved by the Ministry of Energy, Agriculture, the Environmentand Rural Areas of the federal state Schleswig Holstein, Germany[permit number: V312-72241.121-19 (70-6/07)], the Danish Nature

Agency (SNS-3446-00054 and SN 2001-34461/SN-0005) and theAnimal Welfare Division (Ministry of Justice, Denmark, 2005/561-976).

Serumwas taken from a total of 313 free-ranging harbour seals.141 serum samples were taken in June, one sample in May and twosamples in July. As 95% of harbour seals are born in June, andnursing takes four weeks, pups sampled in May and June wereconsidered as not weaned (Ross et al., 1994; Abt, 2002). The twoanimals sampled in July had an unknown weaning status andharbour seals captured in September 2014 were designated asweaned because sampling was conducted when the harbour sealshad already finished nursing.

Because of their approximate age, AG 3 and AG 4 animals wereconsidered as weaned regardless of the sampling date. Detailedinformation on sampled harbour seals is given in Table 1.

Blood was takenwith a 1.20� 100mm needle (SUPRA, EhrhardtMedizinprodukte, Geislingen, Germany) from the extraduralintervertebral sinus 5 cm cranial to the pelvis (Dierauf and Gulland,2001), or from the tarsal sinus of the hind flippers (SanchezContreras, 2014) and filled in tubes containing serum separationgel. Serum was obtained by centrifugation at 3000x g for 15 minand stored at �20 �C until use.

2.1.3. Sera of harbour seals in rehabilitationConsecutive serum samples were taken for routine diagnostic

examinations from six harbour seals at the Seal Rehabilitation andResearch Center (SRRC), Pieterburen, The Netherlands (Table 1).The animals were found between January and June 2015 and weresuspected or diagnosed to have lungworm infections. O. circumlitusspecimens were found in sputum in three of six individuals, and allsix harbour seals showed symptoms like dyspnoe, coughing and apoor body condition. Samples were taken on three to five differentoccasions during a period of about 12e19 weeks (Fig. 1). Blood wassampled and processed as described above.

2.1.4. Statistical analysesStatistical significance of differences between body weight and

body length and age distributionwithin the lungworm infected andnegative animals was tested using SigmaStat (version 3.11, SystatSoftware GmbH, Erkrath, Germany).

2.1.5. ELISAAll sera were tested with the harbour/grey seal adapted re-

combinant MSP-ELISA as previously described (Ulrich et al., 2015).All samples were analysed in duplicates and the optical density(OD) arithmetic mean of the duplicates was corrected for the blankvalue. Serum samples were assigned as positive or negativeconsidering the evaluated cut-off value of 0.422 OD (Ulrich et al.,2015).

2.2. Molecular characterisation of MSP in parasitic nematodes ofharbour seals and harbour porpoises

2.2.1. Parasite materialAdult nematodes were collected during necropsies of harbour

seals and harbour porpoises and their species identified by ste-reomicroscopic examination (45x magnification; Olympus® SZ 61,Hamburg, Germany). Nematodes of harbour seals included lung-worms (O. circumlitus, Crenosomatidae: Metastrongyloidea andP. gymnurus, Filaroididae: Metastrongyloidea), heartworms (Acan-thocheilonema spirocauda, Onchocercidae: Filarioidea), intestinalnematodes (Contracaecum osculatum and Pseudoterranova deci-piens, Anisakidae: Ascaridoidea) and lungworms of harbour por-poises (Pseudalius inflexus, Torynurus convolutus, Halocercusinvaginatus and Stenurus minor, Pseudaliidae: Metastrongyloidea).

Table 1Lungworm seroprevalence in harbour seals.

Harbour seal age group (AG) Origin/coordinates No. of samples (male/female) Seroprevalence % (proportion)

AG 1 free-ranging(not weaneda)

North Sea coast of the federal stateSchleswigeHolstein, Germany/54�48055.500N 8�39010.000E

144 (74 m/70 f) 1 (m)/0.7% (100% m)

AG 2 free-ranging(weaned)

Anholt island in the Kattegat, Denmark/56�42040.900N 11�31006.500E

9 (5 m/4 f) 8 (4 m, 4 f)/88.9% (50.0% m, 50.0% f)

AG 3 free-ranging(weaned)

Lorenzensplate, sandbank off the North Sea coast of the federalstate SchleswigeHolstein, Germany/54�25000.300N 8�29035.700E

28 (18 m/10 f) 15 (10 m, 5 f)/53.7% (66.6% m/33.3% f)

AG 4 free-ranging(weaned)

Lorenzensplate, sandbank off the North Sea coast of the federalstate SchleswigeHolstein, Germany/54�25000.300N 8�29035.700E

132 (72 m/60 f) 32 (17 m, 15 f)/24.2% (53.1% m, 46.9% f)

AG 3 in rehabilitation(weaned)

North Sea coast of The Netherlands/53�25032.200N 6�27020.000E

6 (5 m/1 f) 6 (5 m, 1 f)/100% (83.3% m, 16.7% f)

a Except two individuals with an unknown weaning status.

S.A. Ulrich et al. / International Journal for Parasitology: Parasites and Wildlife 5 (2016) 48e5550

Nematodes were washed with a 0.9% sodium chloride solution andstored at �80 �C until use.

2.2.2. Characterisation of genomic MSP sequencesGenomic DNA was isolated from female and male individuals

using NucleoSpin® Tissue Kit (MachereyeNagel, Dueren, Germany)following the manufacturer's instructions. Degenerated primersspanning the MSP gene from the start to the stop codon weredesigned based on nine known MSP sequences [D. viviparus(GenBank accession no. EF012201), Dictyocaulus arnfieldi(KR267306), Dictyocaulus eckerti (KR267310), Oesophagostomumdentatum (AJ627870), Ancylostoma ceylanicum (CB176474), Hae-monchus contortus (BM138909), Nippostrongylus brasiliensis(BU493394), Teladorsagia circumcincta (CB037984) and Trichos-trongylus vitrinus (AJ616498)]. Selected primer sequences wereMSP deg for: 50-ATGGCHWCAGTTCCHCCHGGHGAYATC-30 and MSPdeg rev: 50-TCADGGRTTGTAYTCRATVGG-30. PCR reaction setup wasas follows: 16 ml deionized H2O, 2.5 ml 10 � buffer, 0.5 ml dNTP(10 mM each), 1.25 ml forward and reverse primer (10 mM each) and0.5 ml Perfect Taq Polymerase (5 U/ml, 5 PRIME GmbH, Hilden,Germany), respectively were added to 3 ml genomic DNA as tem-plate. PCR cycling (40 cycles) was performed using the followingtemperature profile: Initial denaturing at 95 �C for 4 min, dena-turing at 95 �C for 30 s, annealing primers at 55 �C for 1 min,extending primers at 72 �C for 1 min, and final extension at 72 �Cfor 10 min. Gel electrophoresis [1% agarose gels stained with GelRed® (Biotium, Inc., Hayward, Canada)] was performed and visiblebands were cut out of the gel, ligated into pCR® 4-TOPO® vector

Fig. 1. Anthelmintic treatment pattern, blood sampling time points and corresponding OD vivermectin at arrival (day 1) at the Seal Rehabilitation and Research Centre, Pieterburen, Thvalues in grey boxes show lungworm-ELISA positive serum samples, those highlighted in blucoughed up lungworms one day after arrival. (For interpretation of the references to colou

followed by transformation of Escherichia coli One Shot® TOP10 cells (TOPO TA Cloning® Kit for Sequencing; Invitrogen, Karls-ruhe, Germany). Plasmid inserts obtained by using the DNANucleoSpin® Plasmid Kit (MachereyeNagel, Dueren, Germany)were sequenced at the SEQLAB Sequence Laboratories (G€ottingen,Germany). Sequences of the degenerated primers were removedfrom the received MSP sequences before submission to GenBank(accession nos. KR267315-KR267323) and phylogenetic analyses.

2.2.3. Generation of the complete MSP mRNA transcript sequence ofO. circumlitus

To obtain the full-length MSP mRNA transcript, gene-specific 30

and 50 RACE primers were designed based on the obtainedO. circumlitus genomic MSP sequence with the PrimerSelect pro-gram (DNASTAR, version 5.06; GATC Biotech, Konstanz, Germany).Designed 30 RACE primer sequence was 50-ATGCGA-TACGTTTGAGTATGGTCGTGAGGACACCA-30, the 50 RACE primersequence was 50-GTGTTGCACCACTCAACAGTGATACGGTCG-3’. RACEexperiments were performed using the SMART™ RACE cDNAAmplification Kit (Clontech, Heidelberg, Germany) following themanufacturer's recommendations. The full length transcript wasgenerated by overlapping sequences using Clone Manager 9 (Pro-fessional Edition, Scientific and Educational Software, Morrisville,North Carolina, USA) and submitted to GenBank (accession no.KR267324).

2.2.4. Phylogenetic analysesFor phylogenetic analyses, intron sequences were removed from

alues of harbour seals in rehabilitation. Harbour seals (n ¼ 6) were treated twice withe Netherlands, and 21 days later and once with mebendazole between day 2 and 6. ODe lungworm-ELISA negative samples. Harbour seal individuals highlighted in dark greyr in this figure legend, the reader is referred to the web version of this article.)

S.A. Ulrich et al. / International Journal for Parasitology: Parasites and Wildlife 5 (2016) 48e55 51

the genomic MSP sequences to compare coding sections only. MSPnucleotide and deduced amino acid sequences of harbour seal andharbour porpoise nematodes were compared to those of nema-todes affecting terrestrial animals. The sequences were alignedwith the Clustal W method and phylogenetic analyses were per-formed by bootstrap tests of phylogeny (1000 replicates) using themaximum likelihood method of the software package MEGA 6.0(Tamura et al., 2013), respectively. The best fit model was deter-mined comparing maximum likelihood fits of 24 different nucleo-tide substitution models including General Time Reversible (GTR),Hasegawa-Kishino-Yano (HKY), Tamura-Nei (TN93), Tamura 3-parameter (T92), Kimura 2-parameter (K2), Jukes-Cantor (JC) andmaximum likelihood fits of 48 different amino acid substitutionmodels including General Time Reversible (GTR), Jones-Taylor-Thornton (JTT), General Reverse Transcriptase (rtREV), GeneralReversible Chloroplast (cpREV), General Reversible Mitochondrial(mtREV24). Models were determined in consideration of the cor-rected Akaike Information Criterion (AICc), the Bayesian Informa-tion Criterion (BIC), the Maximum Likelihood value (InL) and thenumber of parameters (including branch lengths) by using thesoftware MEGA 6.0 (Nei and Kumar, 2000; Tamura et al., 2013).

3. Results

3.1. ELISA

3.1.1. Sera of free-ranging harbour sealsIn all four age groups of habour seals the lungworm seropre-

valence was 17.9% (56/313 individuals). Separated according to sex,18.9% male (32/169) and 16.7% female (24/144) harbour seals wereseropositive.

In AG 1 only one serum sample of the male harbour seal pupssampled in mid-July (unknown weaning status) showed a positiveELISA result with 0.582 OD (0.7%, Table 1). The arithmetic mean ODvalue of all 144 analysed samples of AG 1 was 0.056 OD (SD 0.075).Within the AG 2 to 4, a lungworm seroprevalence of 32.5% wasdetermined with 55 lungworm-positives out of 169 total samples(Table 1). The arithmetic mean of the positive samples was 0.912OD [standard deviation (SD) ¼ 0.51], whereas the arithmetic meanof the negative samples was 0.219 OD (SD ¼ 0.09). In AG 2, 88.9% ofharbour seals were seropositive, 53.6% in AG3 and 24.2% in AG 4which was significantly less prevalent (P¼<0.0001) than in AG 2and AG 3. A significant decrease in body weight (P ¼ 0.003) andbody length (P¼<0.001) was determined in lungworm-positiveanimals of AG 4 (Table 2). There was no significant difference

Table 2Differences between body weight and body length within age groups.

Lungworm seropositives

AG 2No. of seals 8Mean OD 1.264 (SD 0.72)Mean body weight [kg] 22.6 (SD 2.6)Mean body length [cm] 112.4 (SD 7.6)AG 3No. of seals 15Mean OD 1.019 (SD 0.516)Mean body weight [kg] 35.8 (SD 5.6)Mean body length [cm] 134.3 (SD 11.3)AG 4No. of seals 32Mean OD 0.774 (SD 0.399)Mean body weight [kg] 56.2 (SD 15.5)Mean body length [cm] 151.8 (SD 12.7)

a Not determined because of low sample size.b Statistically significant (P � 0.05).

between male and female harbour seal lungworm infection rates.Detailed results on arithmetic means of the positive and negativeOD values and SD of the different age groups are given in Table 2.

3.1.2. Sera of harbour seals in rehabilitationSerum samples of all six investigated individuals (AG 3) showed

positive OD values on arrival at the Seal Rehabilitation and ResearchCentre, Pieterburen, The Netherlands (arithmetic mean: 1.345 OD;SD 0.512). Three of six animals coughed up lungworms on arrival(individuals 1e3, Fig. 1). One harbour seal showed a negative OD(0.339 OD) seven days after the first antiparasitic treatment, twomore animals turned antibody-negative 60 days after, and a furtherindividual 88 days after the second antiparasitic treatment. Two ofsix individuals still showed positive ODs at the final examinationbefore release 71 and 112 days after finishing the antiparasitictreatment scheme (Fig. 1).

3.2. Molecular characterisation of MSP in harbour seal and harbourporpoise parasitic nematodes

Genomic MSP sequences of the nine investigated marinemammal nematode species contained intron sequences between59 and 83 base pairs (bp) in length. The full-length MSP mRNA ofO. circumlitus included 415 bp without poly(A)þ tail. The 50 un-translated region (UTR) consisted of 14 bp, the 3’ UTR of 17 bp. Thefull coding sequence comprised 381 bp encoding 126 amino acidsas well as D. viviparus. Sequence comparison showed thatO. circumlitus and D. viviparus differed in one amino acid at position34, at which isoleucine of D. viviparus was substituted by methio-nine in O. circumlitus. P. gymnurus differed in three amino acidsfrom D. viviparus, most substitutions (six amino acids) were foundfor P. decipiens (Anisakidae, Ascaridoidea). Alignments are providedin Figs. 2 and 3. Phylogenetic trees including 18 MSP sequences ofmarine and terrestrial mammal parasitic nematodes on nucleotideand amino acid level are given in Fig. 4 and 5, respectively. TheKimura 2- Parameter using a discrete Gamma distributionwith fiverate categories was defined as the best fit nucleotide substitutionmodel with 35 parameters, a BIC of 3519,661638, a AICc of3290,173685 and an InL of �1609,846293. The best fit amino acidsubstitution model was determined to be Jones-Taylor-Thorntonusing a discrete Gamma distribution with five rate categorieswith 34 parameters, a BIC of 1137,728758, a AICc of 951,1837439 andan InL of �440,9376663.

On nucleotide level, D. viviparus, D. arnfieldi and D. eckerti,belonging to the subfamily Dictyocaulidae (Trichostrongylidea),

Lungworm seronegatives P-value

10.32524 n.d.a

116 n.d.a

130.275 (SD 0.089)40 (SD 5.2) 0.051136.2 (SD 11.8) 0.67

1000.211 (SD 0.087)67.5 (SD 16.2) 0.003b

160.9 (SD12.1) <0.001b

Fig. 2. Alignment of MSP nucleotide sequences of marine and terrestrial mammal parasitic nematodes using the Clustal W method.

S.A. Ulrich et al. / International Journal for Parasitology: Parasites and Wildlife 5 (2016) 48e5552

showed a genetic similarity of 94% and 82% (Fig. 4) and were 100%identical on amino acid level (Fig. 5). On amino acid level, MSP ofmarine mammal parasites grouped in the same cluster as the Dic-tyocaulidae (Trichostrongyloidea). The other included species ofthe superfamily Trichostrongyloidea (N. brasiliensis, T. vitrinus, T.circumcincta, H. contortus), together with the two further terrestrialmammal parasites Oesophagostomum dentatum (Strongyloidea)and A. ceylanicum (Ancylostomatoidea), formed a separate clusterfrom the Dictyocaulidae and marine mammal parasites (Fig. 5).

4. Discussion

In the present study, prevalence of lungworm infection in livefree-ranging harbour seal populations was investigated for the firsttime. Assuming lungworm larvae transmission to be dependent onpreying upon the intermediate hosts, unweaned seal pups (AG 1)were unlikely to be infected by O. circumlitus and P. gymnurus as

Fig. 3. Alignment of MSP amino acid sequences of marine and terres

they did not consume fish or other potential intermediate hosts(Dailey, 1970; Bergeron et al., 1997a; Lehnert et al., 2010). Sup-porting that hypothesis, lungworm seroprevalence in AG 1 was0.7%. The one seropositive individual of AG 1 was sampled in mid-July, about six weeks after majority of harbour seals give birth totheir offspring (Bonner, 1972; Siebert et al., 2012). After nursing andthe post-weaning fast, which takes approximately six weeks intotal (Muelbert and Bowen, 1993; Ross et al., 1994), the lungworm-positive specimen may have been already preying and developingadult parasites. Even though a lungworm-infection in mid-Julyseems early, it may be explained by early births during thebreeding period (Measures, 2001). Additionally, the measured ODvalue of the seropositive seal pup is 0.582 OD, which is relativelyclose to the cut-off value of 0.422 OD and distinctively away fromthe mean positive OD values of AG 2 (1.264 OD) and AG 3 (1.019OD). This indicates that the lungwormpositive seal pupwas in earlypatency and had just started to develop anti-MSP antibodies, which

trial mammal parasitic nematodes using the Clustal W method.

94

6152

70

79

78

98

48

94

82

67

36

68

39

0.02

Acanthocheilonema spirocauda (KR267316), Metastrongyloidea

Parafilaroides gymnurus (KR267319), Metastrongyloidea

Pseudoterranova decipiens (KR267318), AscaridoideaContracaecum osculatum (KR267315), Ascaridoidea

Halocercus invaginatus (KR267317), Metastrongyloidea

Stenurus minor (KR267321), Metastrongyloidea

Torynurus convolutus (KR267322), Metastrongyloidea

Pseudalius inflexus (KR267320), Metastrongyloidea

Otostrongylus circumlitus (KR267323), Metastrongyloidea

Dictyocaulus arnfieldi (KR267306), Trichostrongyloidea

Dictyocaulus eckerti (KR267310), TrichostrongyloideaDictyocaulus viviparus (EF012201), Trichostrongyloidea

Oesophagostomum dentatum (AJ627870), Strongyloidea

Ancylostoma ceylanicum (CB176474), Ancylostomatoidea

Haemonchus contortus (BM138909), Trichostrongyloidea

Nippostrongylus brasiliensis (BU493394), Trichostrongyloidea

Teladorsagia circumcincta (CB037984), Trichostrongyloidea

Trichostrongylus vitrinus (AJ616498), Trichostrongyloidea

Fig. 4. Phylogenetic tree from maximum likelihood analysis of MSP nucleotide sequences of marine and terrestrial mammal parasitic nematodes including GenBank accessionnumbers. The percentage of replicate trees in which the associated species clustered together in the bootstrap test (1000 replicates) is shown next to the branches.

S.A. Ulrich et al. / International Journal for Parasitology: Parasites and Wildlife 5 (2016) 48e55 53

supports the aforementioned aspects on infection time pattern.In AGs 2 to 4, 32.5% of the free-ranging harbour seal population

in the German Wadden Sea and Danish Kattegat was seropositivefor lungworm infections. No correlation between sex distributionand lungworm infection was found. The majority of seropositiveanimals belonged to AG 2 (88.9%) and AG 3 (53.6%), covering 7weekse18 months of life, confirming age-related lungworminfection reported previously (Onderka, 1989; Claussen et al., 1991;Lehnert et al., 2007; Siebert et al., 2007). AG 3 represents a tran-sition between young seals (AG 2) being infected often by lung-worms and adult seals (AG 4) showing a seroprevalence of only24.2%.

31

50

74

46

67

0.005

Dictyocaulus eckerti (ALI16159), Trichostrongyloid

Dictyocaulus viviparus (AAB27962), Trichostrongy

Dictyocaulus arnfieldi (ALI16155), Trichostrongylo

Torynurus convolutus (ALI16171), Metastrongyloid

Stenurus minor (ALI16170), Metastrongyloidea

Pseudalius inflexus (ALI16169), Metastrongyloidea

Halocercus invaginatus (ALI16

Otostrongylus circumlitus (ALI16172), Me

Acanthocheilonema spirocauda

Parafilaroides gymnurus (ALI1

Nippostrongylus brasiliensis (BU493394*), Trichostr

Trichostrongylus vitrinus (AJ616498*), TrTeladorsagia circumcincta (CBAncylostoma ceylanicum (CB17

Haemonchus contortus (BM138

Fig. 5. Phylogenetic tree from maximum likelihood analysis of MSP amino acid sequencesnumbers. The percentage of replicate trees in which the associated species clustered togethsequence translated from EST sequences.

In contrast to harbour seals, lungworm infections in harbourporpoises seem to accumulate with age and could possibly haveinfections throughout their life-time (Measures, 2001; Lehnertet al., 2005). This may be due to harbour porpoises being weanedeight to ten months later than harbour seals (Lockyer, 2003). Theytherefore get infected at a later point when they start ingesting preyspecies (Lockyer, 2003). However, transmission of marine lung-worm larvae is not completely understood. Besides food-borneinfection, transplacental transmission in cetaceans has beenhypothesised (Dailey et al., 1991) and transmammary infectioncannot be excluded (Conlogue et al., 1985). Another explanation forparasite accumulation in harbour porpoises is the possible lack of

ea

loidea

idea

ea

166), Metastrongyloidea

tastrongyloidea

Pseudoterranova decipiens (ALI16167), Ascaridoidea

Contracaecum osculatum (ALI16164), Ascaridoidea(ALI16165), Filaroidea

6168), Metastrongyloidea

ongyloidea

ichostrongyloidea037984*), Trichostrongyloidea6474*), Ancylostomatoidea

909*), TrichostrongyloideaOesophagostomum dentatum (CAF29502), Strongyloidea

of marine and terrestrial mammal parasitic nematodes including GenBank accessioner in the bootstrap test (1000 replicates) is shown next to the branches. *amino acid

S.A. Ulrich et al. / International Journal for Parasitology: Parasites and Wildlife 5 (2016) 48e5554

protective immunity to lungworm infections in this marinemammal species.

Because adult harbour seals were observed to be less infectedthan young seals, a development of a protective immunity has beenassumed (Claussen et al., 1991; Bergeron et al., 1997b). Such im-munity has been described in cattle against the bovine lungwormD. viviparus (Jarrett et al., 1958, 1959; Enigk and Hildebrandt, 1969;McKeand et al., 1995), lasting for a period of approximately six totwelve months (Michel et al., 1965). Whether seropositive animalsof AG 4 are due to a reinfection event after loss of immunity or dueto remaining antibodies against a lungworm infection in previousage groups remains unknown. To gain first information on thepersistence of anti-lungworm antibodies in harbour seals, six in-dividuals were monitored serologically during rehabilitation. Ani-mals were treated with anthelmintics after arrival to therehabilitation center, but this may not have an impact on antibodypersistence. For cattle it has been shown that anthelmintic treat-ment in advanced patency did not influence lungworm antibodytiters (Fiedor et al., 2009). All six harbour seals revealed positive ODvalues on arrival. The longest seropositivity determined was for anindividual that was still positive at release 132 days after arrival.Another animal turned seronegative between day 100 (fourthsampling) and day 109 (fifth sampling), and another still, wasseropositive at its release on day 92. The second half of monitoredharbour seals showed a shorter period of antibody persistence. Twoanimals turned seronegative between day 13 (second sampling)and day 81 (third sampling) after arrival, and one individual wasalready seronegative 13 days after arrival. As the latter had a verylow positive OD value on arrival, it may be concluded that this in-dividual was already in the post-patency phase. However, as thetime point of infection and the duration of patency in harbour sealsis unknown (Measures, 2001), it can only be assumed that thoseharbour seals with longer lasting antibody titers got infected at alater point compared to those whose antibodies decreased earlier.Studies on serum persistence of lungworm-antibodies in cattlerevealed positive OD values until day 41e203 post infection (pi)with an overall mean detection period until day 126e143 pi(Cornelissen et al., 1997; Fiedor et al., 2009). Antibody persistencein harbour seals may be similar, but further studies with a largersample size or experimental infection studies are needed toconfirm this presumption.

Harbour seals that had never been affected by lungworms orthose who had overcome a lungworm infection relatively early, areassumed to be able to dive and prey appropriately to their needs asopposed to infected seals. As a consequence, lungworm infectedseals are not able to grow as fast as their healthy conspecifics due torespiratory difficulties and shorter diving and feeding times(Onderka, 1989; Measures, 2001). This became obvious in AG 4, inwhich lungworm seropositive harbour seals showed statisticallysignificantly reduced body weights and lengths compared to thesero-negative individuals. Lungworm infected harbour seals in AG3 showed a trend towards reduced body weight (P ¼ 0.051),whereas body length was unaffected. Similarly, a negative corre-lation between infection with O. circumlitus and sternal blubberthickness has been reported for ringed seals (Bergeron et al.,1997b).

Lungworms, as well as all other nematodes, possess the spermprotein MSP, which enables sperm motility (Miller et al., 2001;Smith, 2014). The molecular characteristics of this protein hasbeen described in several terrestrial host species, including thebovine lungworm D. viviparus (Strube et al., 2009), whose MSPserves as diagnostic antigen in the cattle as well as harbour seallungworm ELISA (vonHoltum et al., 2008; Fiedor et al., 2009; Ulrichet al., 2015). However, no molecular information on MSP fromnematodes infecting harbour seals and harbour porpoises was

available. As MSP is essential for nematode reproduction, and thusfor conservation of the species, conservation of the protein wasexpected (Klass and Hirsh, 1981; Miller et al., 2001; Smith, 2014).The phylogenetic analyses revealed that MSP sequences of the tri-chostrongylid Dictyocaulidae are more closely related to MSP of themetastrongylid lungworms of marine mammals than to trichos-trongylid intestinal nematodes of terrestrial hosts, which formed aseparate cluster with strongylid and ancylostomatid representa-tives. This might indicate that dictyocaulid lungworms may belongto the superfamily Metastrongyloidea rather than to the Trichos-trongyloidea. Phylogenetic analysis of mitochondrial encodedproteins showed that the Dictyocaulidae grouped with the exclu-sion of the terrestrial metastrongylid lungworms of the genusMetastrongylus and Angiostrongylus as well as trichostrongylid(H. contortus), strongylid (O. dentatum) and ancylostomatid (Ancy-lostoma caninum) nematodes (Gasser et al., 2012). As the presentstudy was based on one gene, further analyses are necessary toverify or falsify the current taxonomical allocation of the Dictyo-caulidae to the superfamily Trichostrongyloidea.

5. Conclusions

In all age groups of live free-ranging harbour seals an age-related lungworm seroprevalence of 17.5% was found. Young sealsbetween six weeks and six months were most often infected with aseroprevalence of 88.9% and seals of six to 18 months had a sero-prevalence of 56.5%. Analyses on the persistence of lungworm anti-MSP antibodies in harbour seals showed that a protective immunitymay be assumed and antibody persistence patterns may be similarto that of cattle. The MSP-ELISA proved to be a valuable method toscreen live seals for lungworm infections, providing important datafor health surveillance, management and conservation of free-ranging harbour seal populations. Moreover, the molecular MSPsimilarity within the superfamily Metastrongyloidea provides po-tential for future development of an ELISA for lungworm detectionin harbour porpoises.

Conflict of interest

None of the authors of this paper has a financial or personalrelationship with other people or organisations that could inap-propriately influence or bias the content of the paper.

Acknowledgements

The authors would like thank all helpers at seal catches, thenational park rangers, seal hunters, Danish colleagues and otherhelpers for the collection of harbour seals and J€org Driver for bloodsampling. Parts of the project were funded by the Agency forCoastal Defence National Park and Marine Conservation (ACNM-SH) (4121.5-201-522F) as well as The Ministry of Energy, Agricul-ture, the Environment and Rural Affairs of the federal stateSchleswigeHolstein, Germany (V 5012-0635.70-6).

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